It is known that the reaction of an enzyme with its substrate generates a product. The enzyme is a true catalyst because it is not consumed in the reaction, but is free to generate repetitively more product. The rate at which the product is generated is referred to as the turnover number, a number that varies for different enzymes. In practice, enzymatic activity is most conveniently detected by monitoring a spectrophotometric absorbance of the substrate or product that varies as a result of enzymatic turnover. It is known that some naturally occurring substrates and/or their corresponding products do not possess readily utilizable absorbance peaks, making the spectrophotometric detection of enzymatic activity difficult. In some cases a synthetic substrate can be designed for the detection of enzymatic activity.
Synthetic substrates can be designed to be chromogenic or fluorogenic, i.e., when catalyzed by the enzyme, an optionally detectable change in the substrate and/or product is produced. The most significant limitation to the use of synthetic substrates to detect enzymatic activity is that such substrates cannot always be prepared for the desired enzyme. Detailed knowledge is required of the catalytic properties of each specific enzyme in order to properly design a useful synthetic substrate. For example, synthetic chromogenic substrates for thrombin or Factor X.sub.a have been designed for the detection of thrombin or Factor X.sub.a activity. These synthetic substrates replace the natural substrate, fibrinogen, in chromogenically based assays for these enzymes.
Immunoassays for the detection of analytes are known. The so-called heterogeneous immunoassays can involve an incubation of an antibody with the analyte in the presence of a labeled antigen of substantially identical immunological properties vis a' vis the antibody as the analyte. The amount of labeled antigen bound to the antibody or free in solution is determined after an appropriate separation procedure, as a measure of the amount of analyte present. The first such assay was described by R. S. Yalow and S. A. Berson in 1959 (Nature 184:1648). This assay, called a radioimmunoassay, utilized a radionuclide as the label. The use of enzymes as labels to replace the radionuclide with the latter's self-evident storage, handling and safety problems, was described by van Weemen and Schuurs in 1971 (FEBS Letters, Volume 15, 232 (1971) and U.S. Pat. No. 3,791,932, issued Feb. 12, 1974 on an application filed Jan. 27, 1972). The art has seen numerous variations on the basic theme of van Weemen and Schuurs.
It is known that multiepitopic antigenic substances can agglutinate particles with multiple epitope receptors to produce agglomerates, e.g., influenza virus and sheep erythrocytes, respectively. These agglomerates can be detected using light scattering type measurements. Agglutination-inhibition assays are known for the detection of antigens and haptens in liquid test samples. Typically, the binding of a multivalent antibody to highly refractive particles coated with the antigen or hapten is inhibited in a competitive fashion by the antigen or hapten in the test sample. (See Craig et al., U.S. Pat. No. 4,401,765 issued Aug. 30, 1983.) These assays are generally limited in sensitivity to antigens and haptens in the concentration range of 10.sup.-7 to 10.sup.-9 M.
A continuing problem in immunoassays utilizing enzymes as labels is the inability to detect analytes at concentrations below 10.sup.-10 M in a relatively short period of time, typically thirty minutes or less, preferably ten minutes or less. High turnover number enzymes have been used as labels to maximize signal production. Such enzymes include .beta.-galactosidase, horseradish peroxidase, alkaline phosphatase, glucose oxidase, .beta.-lactamase, and urease. The use of chromogenic substrates with these enzymes can provide sensitivity to about 10.sup.-10 M. The use of fluorogenic substrates could lead to even greater sensitivity. However, all biological samples contain fluorescent material, e.g., porphyrins, which can interfere with the fluorogenic substrate measurements. This disadvantage can be overcome with additional sample processing, specifically, separating the bound from enzyme-labeled complex. Interfering fluorescent materials can be removed during this separation, with the constraint that measurements of only the bound enzyme-labeled complex can be made.
High sensitivity immunoassays using enzyme require an undesirable amount of time for the generation of detectable levels of enzymatically generated chromophore or fluorophore, i.e., roughly thirty to ninety minutes to achieve optimum sensitivity, even with high turnover number enzymes and synthetic substrates.
There is a continuing need in the art for a method to detect low levels of enzymatic activity in a test sample in a short amount of time, typically ten minutes or less. There is also a need for a method to measure enzymatic activity when no readily measurable product results from either natural or synthetic substrates.